Nucleic acid molecules cannot easily cross cell membranes because of their size and hydrophilicity. Delivery has therefore been one of the major challenges for nucleic acid therapeutics, e.g., antisense payloads and RNAi technology. To trigger RNase H activity or RNAi activity following systemic administration, a formulation containing nucleic acid molecules not only must (1) protect the payload from enzymatic and non-enzymatic degradation and (2) provide appropriate biodistribution of the formulation, but also (3) allow cellular uptake or internalization of the formulation and (4) facilitate delivery of the nucleic acid payload to the cytoplasm of the cell. Many formulations that excel in criteria 1 and 2 above are deficient in criteria 3 and 4, and many nucleic acid formulations therefore show excellent biodistribution but fail to knock down the target gene due to lack of systemic delivery and local delivery.
While a number of lipid-based formulations have recently been demonstrated to effect intracellular delivery of nucleic acid payloads to at least certain types of mammalian cells (e.g., mammalian liver cells), the precise proportions and methods of combining lipids, payloads and other components of such formulations can greatly influence the extent to which successful delivery of nucleic acid payloads is achieved. Accordingly, modest changes in the processes employed to obtain such lipid-based formulations have the potential to produce dramatic and surprising differences in delivery efficacy. As such, there is a need to optimize the process by which lipid-based formulations of nucleic acid payloads (and, by extension, anionic agents more generally) are obtained, thereby enhancing the delivery of such therapeutic anionic agents to cells.